CN103008037B - Automatic micro-droplet array screening system using method with pico-liter-scale precision - Google Patents

Automatic micro-droplet array screening system using method with pico-liter-scale precision Download PDF

Info

Publication number
CN103008037B
CN103008037B CN201210589055.1A CN201210589055A CN103008037B CN 103008037 B CN103008037 B CN 103008037B CN 201210589055 A CN201210589055 A CN 201210589055A CN 103008037 B CN103008037 B CN 103008037B
Authority
CN
China
Prior art keywords
sample
capillary
reagent
liquid
oil phase
Prior art date
Application number
CN201210589055.1A
Other languages
Chinese (zh)
Other versions
CN103008037A (en
Inventor
方群
祝莹
张云霞
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Priority to CN201210589055.1A priority Critical patent/CN103008037B/en
Publication of CN103008037A publication Critical patent/CN103008037A/en
Application granted granted Critical
Publication of CN103008037B publication Critical patent/CN103008037B/en

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1016Control of the volume dispensed or introduced
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02Burettes; Pipettes
    • B01L3/021Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0605Metering of fluids
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0642Filling fluids into wells by specific techniques
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0673Handling of plugs of fluid surrounded by immiscible fluid
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0684Venting, avoiding backpressure, avoid gas bubbles
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14Process control and prevention of errors
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14Process control and prevention of errors
    • B01L2200/142Preventing evaporation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0819Microarrays; Biochips
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0896Nanoscaled
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406Moving fluids with specific forces or mechanical means specific forces capillary forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0475Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50853Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates with covers or lids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099Characterised by type of test elements
    • G01N2035/00158Elements containing microarrays, i.e. "biochip"
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027General features of the devices
    • G01N2035/1034Transferring microquantities of liquid
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027General features of the devices
    • G01N2035/1034Transferring microquantities of liquid
    • G01N2035/1039Micropipettes, e.g. microcapillary tubes

Abstract

The invention relates to the field of high-throughput screening and particularly relates to an automatic micro-droplet array screening system using method with pico-liter-scale precision. The using method provided by the invention comprises the following steps of: firstly, using liquid with a low thermal expansion coefficient and filled in a liquid driving system and a capillary tube as carrier liquid, and entirely emptying bubbles inside the capillary tube; secondly, soaking a sampling end of the capillary tube in an oil phase sample immiscible with a water phase sample, extracting a section of oil phase sample into the capillary tube, so as to isolate the water phase sample and the carrier liquid; thirdly, soaking the sampling end of the capillary tube into a sample/reagent storage tube and extracting a certain volume of the water phase sample solution into the capillary tube; and finally, moving the sampling end of the capillary tube into the oil phase sample above micro-pores of a micro-porous array chip, and pushing the sample solution in the capillary tube into the micro-pores, so as to form sample droplets. With the adoption of the using method provided by the invention, the liquid is quantitatively measured, and the generated droplets have the pico-liter-scale volume precision, so that the consumption of the samples/reagents in the high-throughput screening can be effectively reduced, and the experiment cost can be saved.

Description

A kind of using method with the automation microlayer model array screening system of skin upgrading precision

Technical field

The field that the present invention relates to is high flux screening field, particularly a kind of using method with the automation microlayer model array screening system of skin upgrading precision.

Background technology

High throughput screening system originates from the research of drug screening, it mainly with 96 or 384 orifice plates for array reactor, Liquid distribution and sample mixed is carried out by automatic machinery people, highly sensitive, checkout gear at a high speed and data processing software is adopted to carry out treatment and analysis to experimental result, realize the screening flux of sample every day at least 10,000.The screening powerful due to it and analytic function, the range of application of High Throughput Screening Assay expands to multiple scientific domains such as biology, medical science, chemistry from drug screening.But, along with increasing sharply of new target and sample size, face huge challenge gradually based on the commercialization high flux liquid handling of porous plate and screening system.Compound for screening is mainly derived from Prof. Du Yucang or separation and purification obtains from natural products, and cost is higher.At present, the sample based on the high throughput screening system of porous plate consumes volume at 1-100 microlitre.If can skin rise to receive upgrading level on handle liquid, sample is screened, then likely its cost is reduced 1000-100000 doubly.Therefore, no matter be in industrial quarters or in academia, large quantifier elimination all concentrates on the microminiaturization of high throughput screening system.Such as, the minimum liquid handling volume of Britain Douglas company development OryxNano series of fluids treating apparatus 100 receives liter (a http://www.douglas.co.uk/oryxnano.htm).The minimum liquid of the Mosquito series of fluids treating apparatus of TTP LabTech company of Britain development is handled volume and is reached 25 and receive liter (www.ttplabtech.com/products/mosquito/).The application of these instruments reduces the cost of screening and research and development significantly.But, still lack at present can several receive to rise even skin upgrading volume carries out liquid and handles and the technology of high flux screening and device.

The Research Challenges of skin upgrading high flux screening is mainly reflected in: 1) existing instrument and equipment is difficult to carry out reliable liquid manipulation in skin upgrading, such as accurate liquid measuring, liquid transfer, sample mixed etc.; 2) reducing along with liquid volume, evaporative effect significantly increases.Such as under typical laboratory condition, the aqueous phase droplets of a skin upgrading can volatilize completely in 1 second; 3) liquid under microbody system has great specific area, due to the molecular self-assembling on water/gas interface and water/liquid/solid interface or nonspecific action, easily cause the inactivation of bioactive molecule, loss and cross pollution, thus cause false positive or the false negative of the selection result.

Based on one of focus that the microflow control technique of drop is in recent years in the microminiaturized research field of high flux screening.It, by the control of heterogeneous fluid in micro scale channel, realizes the generation of Water-In-Oil in enormous quantities or oil-in-water type drop microreactor, sample mixed, reaction and Analysis and Identification.The size of drop reactor can receive upgrading flexible modulation flying to upgrade to, thus may realize super low consumed high flux screening.Due to parcel and the protective effect of oil phase, effectively inhibit solvent evaporation and the diluting effect of drop reactor, and the cross pollution of sample room.Utilize the self assembly effect of bio-compatibility surfactant molecule, can react the microenvironment providing a gentleness homogeneous to biochemical screening, be conducive to the accuracy improving Analysis and Screening.Meanwhile, the volume that drop reactor is small is conducive to the mass transfer between acceleransstoff, improves reaction efficiency.Therefore, the microflow control technique based on drop likely becomes the High Throughput Screening Assay of a new generation because of the characteristic of its excellence.

At present, the micro-fluidic screening technique based on drop mainly contains three kinds, is respectively drop holder method, slip chip method and drop assemble method.Drop holder first adopts the method extracted one by one to be loaded in capillary by different sample solutions to be screened to form drop, then by this capillary and micro-fluidic chip expanding channels, by the T-shaped interface on chip, target solution is injected into triggering reaction in drop, finally the drop reactor of formation is collected in another one capillary and carries out incubation reaction and detection (Zheng B., Ismagilov R. F. Angew. Chem., Int. Ed., 2005,44,2520).First slip chip method then loads sample to be screened and forms drop in the array micropore of bottom sheet, then slide upper slice make the droplets mixing of target reagent solution in a microchannel and bottom sheet, carry out triggering reaction and screening (Du W. B., Li L., Nichols K. P., Ismagilov R. F. Lab Chip, 2009,9,2286).But above-mentioned two kinds of methods all need the connection of manual drop loading, capillary and chip channel, and the chip slide of precision, are difficult to the screening being applied to extensive sample.Drop assemble method utilizes the quick switching of the sample cell of automation and Reagent Tube, the mixing of sample to be screened and reagent is completed in the process that drop is formed, then drop is stored on capillary and chip and carries out reacting and detect (Du W. B., Sun M., Gu S. Q., Zhu Y., Fang Q. Anal. Chem., 2010,82,9941; Fang Qun, Du Wenbin, Sun Meng, based on Microfluidic droplet generation system and the using method of drop der group packing technique, Chinese invention patent, application number: 201010250945.0).Although drop der group packing technique solves the automation of drop screening and a difficult problem for extensive sample screening, the generation due to the drop containing sample and reagent have employed assemble method one by one, and its breakneck acceleration is difficult to improve.In addition, due to the dimensional effect that microminiaturization brings, above-mentioned several drop screening technique is all difficult to carry out biochemical screening mensuration at skin upgrading volume.

In Microfluidic droplet system, the Reagent adding methods of parallelization mainly can be divided into two classes.In the different drop that the first kind adopts a T-shaped branched bottom to be injected into by one identical reagent in main channel (Zheng B., Ismagilov R. F. Angew. Chem., Int. Ed., 2005,44,2520).These class methods often carry out coupling, for the microscreen based on drop with above-mentioned drop holder method.But the subject matter of these class methods there is larger droplet sample at T-shaped passage intersection to remain, thus cause the cross pollution between drop.The reagent that other class methods adopt droplet coalescence technology to carry out parallelization adds.First be in microchannel, generate the sample of pairing and the drop of reagent mutually, then the method that fluid dynamic is assisted or dielectric is assisted is utilized to make the good droplet coalescence of pairing form independently microreactor (Teh S Y, Lin R. Hung L. H., Lee A. P., Lab Chip, 2008,8:198).But this class methods structure is comparatively complicated, difficulty of processing is large, and is difficult to use in the screening system with a large amount of different sample.Further, the realization of above-mentioned two class methods all needs the complicated manual adjustments means such as flow rate of liquid adjustment, drop frequency, the control of droplet size, the feedback of droplet position, is difficult to realize reliable automation, in instrument industrialization, there is larger difficulty.

Summary of the invention

The object of this invention is to provide a kind of automation microarray drop screening system and the using method thereof with skin upgrading resolution ratio.This system can carry out full automatic skin upgrading the measuring of liquid, the formation of different sample drop array, the parallelization of target reagent quantitatively add and reaction assay under micro-scale volume.This system both can be used for high-flux medicaments sifting, also can be used in catalyst screening, enzyme kinetic analysis, medical diagnosis on disease and unicellular and single molecule analysis.

Concrete technical scheme of the present invention is as follows:

The present invention is the using method of the microlayer model array screening system with skin upgrading precision, and system comprises capillary, liquid driven system, microwell array chip, sample/reagent memotron and auto-translating platform, and concrete steps are:

1) liquid of low thermal coefficient of expansion will be full of in liquid driven system and capillary as current-carrying, and be emptied completely the bubble in capillary;

2) capillary sampling end is immersed and extracts one section of oil phase in capillary, for isolating aqueous sample and current-carrying in the immiscible oil phase of aqueous sample;

3) capillary sampling end is immersed the aqueous sample solution extracting certain volume in sample/reagent memotron and enter capillary;

4) by the oil phase above the micropore of capillary sampling end immigration microwell array chip, the sample solution in capillary is pushed out in micropore the drop forming sample;

Following concrete steps are also comprised after step 4) of the present invention:

A) on microwell array chip, form the drop of chemical composition or the different sample multiple to be screened of concentration;

B) in capillary, once extract a large amount of reagent, and capillary sampling end is inserted in the drop of each sample respectively, inject the reagent of certain volume, form drop reactor, complete the mixing of sample and reagent, reaction, detection and screening.

Following concrete steps are also comprised after step 4) of the present invention:

M) on microwell array chip, form the drop of a large amount of reagent;

N) in the drop of each reagent, inject sample solution to be screened respectively, form drop reactor, complete the mixing of reagent and sample, reaction, detection and screening.

Liquid driven system of the present invention has the liquid driven precision that number is received liter/min, and it drives the flow rates of liquid to be 1 to receive liter/min to 500 and to receive liter/min.

The present invention is in order to eliminate liquid driven system when changing liquid driven direction, namely release from extracting to be converted to or extract mechanical return difference existing during this from releasing to be converted to, ensure the liquid measuring precision of skin upgrading, the oil phase first extracting certain additional volumes before extracting aqueous sample or reagent solution enters capillary; When sample or reagent solution are released capillary, also the oil phase additionally extracted is released capillary in the lump.

The liquid of low thermal coefficient of expansion is full of as current-carrying in liquid driven system of the present invention and capillary, prevent in experimentation that temperature fluctuation is on the impact of liquid driven precision, the thermal coefficient of expansion scope of current-carrying is 0.00001/ degree Celsius to 0.0005/ degree Celsius.

The tube wall that capillary employing of the present invention is thinner, is conducive to the liquid measuring and reduction liquid the remaining at capillary sampling end that realize skin upgrading; The pipe thickness scope of capillary is 1 micron to 100 microns.

The using method with the microlayer model array screening system of skin upgrading precision of the present invention, before the use, carries out degassed process to liquid carrier fluid and oil phase, prevents from producing bubble in liquid driven process.

The present invention in use, the top of the micropore of microwell array chip, all cover one deck and the immiscible oil phase of aqueous phase with the top of sample/reagent memotron, expose to prevent micro-drop, sample and reagent and volatilize in atmosphere or polluted, the thickness range of oil phase is 0.1 millimeter to 10 millimeters.

The present invention in use, in order to eliminate the interference that in screening course of reaction, oil phase reacts micro biochemical, the surfactant of bio-compatibility is added in oil phase, utilize surfactant molecule in the self assembly effect of oil-water interfaces, reduce the absorption of biomolecule at interface and inactivation, the concentration of surfactant is 0.01% to 10%.

Advantage of the present invention is mainly: (1) liquid quantitatively measure the volume accuracy with drop formation with skin upgrading, effectively reduce the sample/reagent consumption in high flux screening, save experimental cost; (2) adopt capillary is inserted in sample drop to be screened, inject reagent respectively continuously and complete the mixing of sample and reagent, reaction detection and screening, effectively improve the flux of screening, and reduce the risk producing cross pollution; (3) the process full automation such as liquid measuring, release, drop formation and reagent injection, effectively reduces human error and error, is easy to the industrialization of the system that realizes and extensively universal.

Accompanying drawing explanation

Fig. 1 is droplet array screening system and the using method schematic diagram with skin upgrading precision;

Fig. 2 is the method adopting capillary to insert drop, injects the process schematic of reagent in sample drop to be screened successively;

Fig. 3 be utilize droplet array screening system to carry out Caspase-1 inhibitor sifting overlook fluorescence picture;

Fig. 4 carries out 503nhibiting concentration measurement result record figure to screening the inhibitor 28 obtained.

In figure: 1-capillary, 2-liquid driven system, 3-microwell array chip, 4-liquid carrier fluid, 5-aqueous sample, 6-oil phase, 7-sample/reagent memotron, 8-micropore, the drop of 9-sample, 10-reagent, 11-drop reactor.

Detailed description of the invention

below technical scheme of the present invention is elaborated:

the present invention relates to a kind of microarray drop screening system with skin upgrading resolution ratio, be made up of capillary, liquid driven system, microwell array chip, liquid driven system, sample/reagent memotron and auto-translating platform.Liquid driven system is connected for the quantitative extraction of micro liquid and release with capillary, sample/reagent memotron and microwell array chip are fixed on can on the auto-translating platform of three-dimensional movement, sample/reagent memotron is for storing sample needed for experiment and reagent, and microwell array chip is used for the storage of micro-drop, reaction, detection and screening.

According to the present invention, the described microlayer model array screening system with skin upgrading precision, the using method of its feature is: first will be full of the liquid of low thermal coefficient of expansion in liquid driven system and capillary as current-carrying, and be emptied completely the bubble in capillary; Then capillary sampling end is immersed and extracts one section of oil phase in capillary, for isolating aqueous sample and current-carrying in the immiscible oil phase of aqueous sample; Again capillary sampling end is immersed the aqueous sample solution extracting certain volume in sample/reagent memotron and enter capillary; Finally by the oil phase above the micropore of capillary sampling end immigration microwell array chip, the sample solution in capillary is pushed out in micropore and forms sample drop.

According to the present invention, in order to improve the flux of screening, first on microwell array chip, form chemical composition or the different sample drop multiple to be screened of concentration, then in capillary, a large amount of reagent is once extracted, and capillary sampling end is inserted in each sample drop respectively, inject the reagent of certain volume, form drop reactor, complete the mixing of sample and reagent, reaction, detection and screening.Alternatively scheme, also a large amount of identical chemical compositions and the reagent droplet of concentration can first be formed on microwell array chip, then in the drop of each reagent, inject different chemical composition to be screened and the sample solution of concentration respectively, form drop reactor, complete the mixing of reagent and sample, reaction, detection and screening.

According to the present invention, it is characterized in that, liquid driven system has the liquid driven ability that forward is released and oppositely extracted, flow velocity be 1 skin liter/min to 100 mul/min, the volume measuring liquid rises to 100 microlitres at 1 skin.As preferably, for completing measuring of skin upgrading liquid, described liquid driven system has the liquid driven precision that number is received liter/min, and it drives the flow rates of liquid to be 1 to receive liter/min to 500 and to receive liter/min, and the volume range measuring liquid rises to 1000 skin liters at 1 skin;

According to the present invention, in order to eliminate the liquid driven system mechanical return difference that (when being converted to release state from extraction or when being converted to extraction state from release) exists when changing liquid driven direction, ensure the liquid measuring precision of skin upgrading, the oil phase first extracting certain additional volumes before extracting aqueous sample or reagent solution enters capillary; When sample or reagent solution are released capillary, also the oil phase additionally extracted is released capillary in the lump.

According to the present invention, the liquid of low thermal coefficient of expansion is full of as current-carrying in liquid driven system and capillary, to prevent in experimentation temperature fluctuation on the impact of liquid driven precision, as preferably, the thermal coefficient of expansion scope of current-carrying is 0.00001/ degree Celsius to 0.0005/ degree Celsius.

According to the present invention, for realizing the liquid measuring of skin upgrading and reducing liquid remaining at capillary sampling end, draw sharp process to reduce the most advanced and sophisticated diameter of sampling end and cross-sectional area to the sampling end of capillary, as preferably, sample and hold most advanced and sophisticated diameter range to be 1 micron to 100 microns; With sampling end outer wall, hydrophobization process is carried out to the inwall of capillary simultaneously.

According to the present invention, the capillary that employing tube wall is thinner, is conducive to the liquid measuring and reduction liquid the remaining at capillary sampling end that realize skin upgrading, reduces the cross pollution produced when measuring different liquids.As preferably, the pipe thickness scope of capillary is 1 micron to 100 microns.

According to the present invention, before the use, (vacuum or ultrasonic) degassed process is carried out to liquid carrier fluid and oil phase, prevents from producing bubble in liquid driven process.The existence of bubble significantly can reduce and measures precision to micro-skin upgrading liquid.

According to the present invention, before draw samples (or reagent) solution, extract one section in capillary in advance with the immiscible oil phase of sample (or reagent) solution to isolate the liquid carrier fluid of sample (or reagent) and low thermal coefficient of expansion; As preferably, the length range of oil phase is 50 microns to 20 millimeters.

According to the present invention, microwell array chip is processed multiple pitting for containing micro liquid.The volume range of each pitting is that 1 skin rises to 100 microlitres.

According to the present invention, during use, the sample/Reagent Tube of the microwell array on chip and sample/reagent memotron covers one deck oil phase, volatilize in atmosphere to prevent micro-drop and sample from exposing or polluted, the thickness of oil film is 0.1 millimeter to 10 millimeters.During use, the top of the micropore on microwell array chip, with the top of specimen sample/reagent memotron, all cover one deck and the immiscible oil phase of aqueous phase, expose to prevent micro-drop, sample and reagent and volatilize in atmosphere or polluted, as preferably, the thickness range of oil phase is 0.1 millimeter to 10 millimeters.

According to the present invention, as a kind of preferred version, in order to eliminate screening course of reaction in oil phase to the interference of biochemical reaction, the surfactant of bio-compatibility is added in oil phase, utilize surfactant molecule in the self assembly effect of oil-water interfaces, reduce the absorption of biomolecule at interface and inactivation, as preferably, the concentration range of surfactant is 0.01% to 10%.

According to the present invention, adopt multiple capillary or multiple liquid driving device, carry out the measuring of plurality of liquid sample/reagent simultaneously, release and drop formation operation.

Below in conjunction with specific embodiment, technical scheme of the present invention is described further:

With reference to accompanying drawing, below will describe in detail according to a preferred embodiment of the invention.

Fig. 1 is droplet array screening system and the using method schematic diagram with skin upgrading precision.Its using method is as follows: use capillary 1 as sample probe, be connected by its afterbody with liquid driven system 2.All be full of the liquid of low thermal coefficient of expansion as current-carrying 4 in capillary 1 and liquid driven system 2, and separate aqueous sample 5 and current-carrying 4 in the sampling end introducing one section of capillary 1 with the immiscible oil phase 6 of aqueous phase.Mobile capillary 1 or sample/reagent memotron 7, make capillary 1 sample the oil phase 6 extracting certain additional volumes in end immersion oil phase 6 and enter capillary 1 sampling end; Again capillary 1 is sampled the sample 5 quantitatively extracting certain volume in end immersion sample 5 and enter capillary.And then mobile capillary 1 or microwell array chip 3, make capillary 1 sample the oil phase 6 held and be placed on chip 3 above micropore 8.Micro liquid sample 5 in capillary 1 and the extra oil phase 6 extracted are pushed out in micropore 8 by start liquid drive system 2, form the drop 9 of micro-aqueous sample 5.

Before utilizing this system to generate droplet array, the sampling end of capillary 1 carries out drawing point process, and carries out hydrophobizing surface process to the inside and outside wall of capillary 1 and the surface of microwell array chip 3.Vacuum outgas or ultrasonic degas process are carried out to liquid carrier fluid 4 and oil phase 6, prevents from producing bubble in liquid driven process.

Fig. 2 is the method adopting capillary to insert drop, injects the process schematic of reagent in sample drop to be screened successively.Method according to Fig. 1, microwell array chip 3 generates the array of the sample drop 9 containing different chemical composition or concentration.The reagent 10 of a large amount of volume is extracted in sampling capillary 1, then capillary 1 or microwell array chip 3 is moved, making capillary 1 sample end inserts in sample drop 9, start liquid drive system 2, the reagent 10 releasing certain volume enters in sample drop 9, complete the mixing of sample/reagent, form drop reactor 11.

Embodiment 1

Fig. 3 is droplet array screening system according to Fig. 1 and Fig. 2 and using method, using 32 small-molecule chemical things as sample to be screened, carries out the fluorescence picture that inhibitor sifting obtains using Caspase-1 enzyme as Screening target.First the micromolecular compound of 100 μMs is loaded in sample/reagent memotron.Adopt the droplet array generation method described in Fig. 1 on microwell array chip, generate the drop of 32 small-molecule chemical things, the volume of each drop is 180 skin liters.Then according to the target reagent method for implanting described in Fig. 2, substrate (Z-YVAD-R110) solution (20 μMs) risen respectively to the Caspase-1 enzyme solutions (6 mU/ μ L) and 180 skins that inject 180 skins liters in each drop triggers reaction.Microwell array chip is hatched 1 hour under 35 degrees celsius, adopts the method for fluorescence imaging to obtain experimental result.Analysis design mothod result, the rejection ability of compound to Caspase-1 enzyme is stronger, and enzymatic activity is just weak, and under the same reaction conditions, the speed of catalytic substrate reaction is just low, and in corresponding diagram 3, fluorescence signal is more weak.Therefore, being numbered 28,30 and 32 compounds is the inhibitor that Screening and Identification goes out.

Embodiment 2

Fig. 4 is that No. 28 compounds obtained the screening experiment described in Fig. 3 carry out Caspase-1 enzyme 503nhibiting concentration measurement result record figure.First in sample/reagent memotron, be respectively charged into No. 28 compound solutions that concentration is 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μMs and 100 μMs.Droplet array screening system according to Fig. 1 and Fig. 2 and using method, microwell array chip generates the drop (180 skin liter) containing variable concentrations No. 28 compound solutions, then substrate (Z-YVAD-R110) solution (20 μMs) of the Caspase-1 enzyme solutions (6 mU/ μ L) and 180 skins liter that inject 180 skins liters in each drop triggers reaction.Obtain result fluorescence picture according to the method for embodiment 1, fluorescent brightness value is extracted and is normalized.The concentration of compound 28 is taken the logarithm process, and the normalization fluorescent brightness value processing the result that obtains corresponding with concentration is mapped.Utilize data processing software to carry out Sigmoidal matching to result figure, obtaining 503nhibiting concentration is 31.6 ± 3.4 nM.

Claims (10)

1. one kind has the using method of the microlayer model array screening system of skin upgrading precision, described system comprises capillary (1), liquid driven system (2), microwell array chip (3), sample/reagent memotron (7) and auto-translating platform, it is characterized in that, concrete steps are:
1) liquid of low thermal coefficient of expansion will be full of in liquid driven system (2) and capillary (1) as current-carrying (4), and be emptied completely the bubble in capillary (1);
2) immersion of capillary (1) sampling end is extracted one section of oil phase (6) in capillary (1) with (6) in aqueous sample (5) or reagent (10) immiscible oil phase, for aqueous sample (5) or reagent (10) being isolated with current-carrying (4);
3) in order to eliminate liquid driven system (2) when changing liquid driven direction, namely release from extracting to be converted to or extract mechanical return difference existing during this from releasing to be converted to, ensure the liquid measuring precision of skin upgrading, the oil phase (6) first extracting certain additional volumes before extracting aqueous sample (5) or reagent (10) solution enters capillary (1);
4) capillary sampling end is immersed aqueous sample (5) solution extracting certain volume in sample/reagent memotron (7) and enter capillary (1);
5) capillary (1) sampling end is moved in the oil phase (6) of micropore (8) top of microwell array chip (3), sample (5) solution in capillary (1) is pushed out to the drop (9) forming sample (5) in micropore (8), the oil phase (6) extracted extra in step 3) is released capillary (1) in the lump simultaneously.
2. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1, is characterized in that, also comprise following concrete steps after described step 5):
A) at the upper drop (9) forming chemical composition or the different sample multiple to be screened of concentration of microwell array chip (3);
B) in capillary (1), a large amount of reagent (10) is once extracted, and capillary (1) sampling end is inserted in the drop (9) of each sample respectively, inject the reagent (10) of certain volume, form drop reactor (11), complete the mixing of sample (5) and reagent (10), reaction, detection and screening.
3. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1, is characterized in that, also comprise following concrete steps after described step 5):
M) at the upper drop forming a large amount of reagent (10) of microwell array chip (3);
N) in the drop of each reagent (10), inject sample (5) solution to be screened respectively, form drop reactor (11), complete the mixing of reagent (10) and sample (5), reaction, detection and screening.
4. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1 or 2 or 3, it is characterized in that, described liquid driven system (2) has the liquid driven precision that number is received liter/min, and it drives the flow rates of liquid to be 1 to receive liter/min to 500 and to receive liter/min.
5. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1 or 2 or 3, it is characterized in that, the liquid of low thermal coefficient of expansion is full of as current-carrying (4) in described liquid driven system (2) and capillary (1), prevent in experimentation that temperature fluctuation is on the impact of liquid driven precision, the thermal coefficient of expansion scope of described current-carrying (4) is 0.00001/ degree Celsius to 0.0005/ degree Celsius.
6. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1 or 2 or 3, it is characterized in that, the length for the oil phase (6) of aqueous sample (5) or reagent (10) being isolated with current-carrying (4) is 50 microns to 20 millimeters.
7. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1 or 2 or 3, it is characterized in that, described capillary (1) employing tube wall is thinner, is conducive to the liquid measuring and reduction liquid the remaining at capillary (1) sampling end that realize skin upgrading; The pipe thickness scope of described capillary (1) is 1 micron to 100 microns.
8. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1 or 2 or 3, it is characterized in that, before the use, degassed process is carried out to liquid carrier fluid (4) and oil phase (6), prevent from producing bubble in liquid driven process.
9. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1 or 2 or 3, it is characterized in that, during use, the top of the micropore (8) of microwell array chip (3), one deck and the immiscible oil phase of aqueous phase (6) is all covered with the top of sample/reagent memotron (7), expose to prevent micro-drop, sample and reagent and volatilize in atmosphere or polluted, the thickness range of described oil phase (6) is 0.1 millimeter to 10 millimeters.
10. the using method with the microlayer model array screening system of skin upgrading precision according to claim 1 or 2 or 3, it is characterized in that, during use, in order to eliminate the interference that in screening course of reaction, oil phase (6) reacts micro biochemical, the surfactant of bio-compatibility is added in oil phase (6), utilize surfactant molecule in the self assembly effect of oil-water interfaces, reduce the absorption of biomolecule at interface and inactivation, the concentration of surfactant is 0.01% to 10%.
CN201210589055.1A 2012-12-31 2012-12-31 Automatic micro-droplet array screening system using method with pico-liter-scale precision CN103008037B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201210589055.1A CN103008037B (en) 2012-12-31 2012-12-31 Automatic micro-droplet array screening system using method with pico-liter-scale precision

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN201210589055.1A CN103008037B (en) 2012-12-31 2012-12-31 Automatic micro-droplet array screening system using method with pico-liter-scale precision
US14/758,482 US9804185B2 (en) 2012-12-31 2013-11-08 Application method for automatic micro droplet array screening system with picoliter scale precision
PCT/CN2013/086729 WO2014101575A1 (en) 2012-12-31 2013-11-08 Application method for automatic micro-droplet array screening system with picoliter precision

Publications (2)

Publication Number Publication Date
CN103008037A CN103008037A (en) 2013-04-03
CN103008037B true CN103008037B (en) 2015-04-01

Family

ID=47957467

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201210589055.1A CN103008037B (en) 2012-12-31 2012-12-31 Automatic micro-droplet array screening system using method with pico-liter-scale precision

Country Status (3)

Country Link
US (1) US9804185B2 (en)
CN (1) CN103008037B (en)
WO (1) WO2014101575A1 (en)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103008037B (en) 2012-12-31 2015-04-01 浙江大学 Automatic micro-droplet array screening system using method with pico-liter-scale precision
CN103698382A (en) * 2013-12-31 2014-04-02 浙江大学 Capillary electrophoresis analysis device for trace droplet array and application method thereof
CN103954786B (en) * 2014-04-21 2015-07-08 浙江大学 Semi-contact under-oil continuous droplet sample applying and liquid adding method
CN104492508B (en) * 2014-11-13 2016-08-24 浙江大学 A kind of ultramicron liquid drop control device and method based on liquid residue
WO2016078340A1 (en) 2014-11-17 2016-05-26 中国科学院微生物研究所 Apparatus, system, and method for dispensing/mixing a small quantity of liquid
CN104849111B (en) * 2015-04-14 2018-04-20 浙江大学 The forming method of gradient microlayer model array based on sequential injection and microflow control technique
CN106540757B (en) * 2015-09-21 2018-10-26 中国科学院大连化学物理研究所 A kind of magnetic drive liquid quantitative control device
GB201518392D0 (en) * 2015-10-16 2015-12-02 Isis Innovation Microfluidic arrangements
CN107422059A (en) * 2017-03-27 2017-12-01 浙江大学 A kind of device and its application method for submicrosample original position chromatography column feed materials

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6203759B1 (en) * 1996-05-31 2001-03-20 Packard Instrument Company Microvolume liquid handling system
DE10017791A1 (en) * 2000-04-10 2001-10-11 Basf Ag Method and device for microdosing small amounts of liquid for biopolymer arrays
DE10052819B4 (en) * 2000-10-24 2004-02-19 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Pipette system and pipette array and method for filling a pipette system
US6734424B2 (en) * 2002-05-16 2004-05-11 Large Scale Proteomics Corporation Method for microdispensing of fluids from a pipette
CN101151370A (en) * 2005-03-29 2008-03-26 株式会社岛津制作所 Reaction vessel, reaction vessel processing apparatus and diagnostic apparatus
CN1818662A (en) * 2006-03-14 2006-08-16 浙江大学 Sequential injection analyzer based on capillaries and utilization thereof
EP1905513A1 (en) * 2006-09-13 2008-04-02 Institut Curie Methods and devices for sampling fluids
CN103153466B (en) * 2010-07-22 2016-04-13 基因细胞生物系统有限公司 Composite fluid pond
CN101957383A (en) * 2010-08-10 2011-01-26 浙江大学 Micro-fluid control liquid drop generation system based on liquid drop sequence assembly technology and use method
WO2012100205A2 (en) * 2011-01-21 2012-07-26 Biodot, Inc. Piezoelectric dispenser with a longitudinal transducer and replaceable capillary tube
JP2013007579A (en) * 2011-06-22 2013-01-10 Seiko Epson Corp Dispensation method
CN102553665B (en) * 2011-11-04 2014-04-02 浙江大学 Microfluidic concentration gradient droplet generating chip, generating device and application
EP2719461A1 (en) * 2012-10-12 2014-04-16 Roche Diagnostics GmbH Pipetting unit and method of pipetting a test liquid
CN103008037B (en) * 2012-12-31 2015-04-01 浙江大学 Automatic micro-droplet array screening system using method with pico-liter-scale precision

Also Published As

Publication number Publication date
US20160202281A1 (en) 2016-07-14
CN103008037A (en) 2013-04-03
US9804185B2 (en) 2017-10-31
WO2014101575A1 (en) 2014-07-03

Similar Documents

Publication Publication Date Title
Du et al. Microfluidics for cell-based high throughput screening platforms—A review
US10543485B2 (en) Slip chip device and methods
Yeh et al. Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip
Dincer et al. Multiplexed point-of-care testing–xPOCT
US10407709B2 (en) Method and device for isolating cells from heterogeneous solution using microfluidic trapping vortices
JP2017060503A (en) Synthetic liquid cell
US10239057B2 (en) Microfluidic devices and methods for cell analysis and molecular diagnostics
AU2015249293B2 (en) DEP force control and electrowetting control in different sections of the same microfluidic apparatus
US9086371B2 (en) Fluidics devices
Livak-Dahl et al. Microfluidic chemical analysis systems
JP6155419B2 (en) Droplet transport system for detection
US9897596B2 (en) Microfluidic disc for use in with bead-based immunoassays
CN102671729B (en) Micro flow control chip used for multi-index biochemical detection
US6409832B2 (en) Protein crystallization in microfluidic structures
ES2271330T3 (en) Microfluidic chemical testing device and procedure.
EP0639223B1 (en) Microfabricated sperm handling devices
JP4230816B2 (en) Plate that automatically stores part of the sample
EP2061598B1 (en) Method for sampling flowable materials
Tachi et al. Simultaneous separation, metering, and dilution of plasma from human whole blood in a microfluidic system
JP5266208B2 (en) Droplet treatment system
CA2479452C (en) Method and device for determining analytes in a liquid
US7939021B2 (en) Droplet actuator analyzer with cartridge
DE69628016T2 (en) Miniaturized differential extraction device and method
US8231844B2 (en) Method and device for manipulating liquids in microfluidic systems
US7482585B2 (en) Testing chip and micro integrated analysis system

Legal Events

Date Code Title Description
PB01 Publication
C06 Publication
SE01 Entry into force of request for substantive examination
C10 Entry into substantive examination
GR01 Patent grant
C14 Grant of patent or utility model